Methods for detecting and treating pain using brain activity

ABSTRACT

Disclosed are methods for detecting pain in a subject, such as a mammal (e.g., a human), using brain activity, e.g., as determined by electroencephalography. The methods are useful for treating or reducing the likelihood of pain in a subject by determining power amplitude from the power spectral density of the waveforms and, e.g., administering a therapeutic agent to the subject. The methods disclosed herein may also be utilized to screen for a therapeutic agent that decreases power amplitude using a non-human animal subject. The methods also feature the stimulation of thalamic reticular nucleus of a subject to treat or reduce pain.

FIELD OF THE INVENTION

The invention features methods for detecting pain using brain activityas determined by, e.g., electroencephalography (EEG), particularly forthe diagnosis and treatment of pain and for screening of therapeuticagents that treat or prevent pain.

BACKGROUND

Rhythmic activity in field potentials, referred to as oscillation, is anessential mode of communication between neuronal ensembles. Recordingsof brain activity invariably feature oscillation at multiplefrequencies. Oscillation in the brain is thought to require corticallayer hierarchy and recruitment of subcortical structures, such as thethalamus. Neuronal oscillation plays a crucial, though as of yetincompletely defined, role in health and disorders of thought andcognition, such as autism and schizophrenia. For instance, painmodulates brain oscillation in animals, including humans. Recent studiesusing functional magnetic resonance imaging (fMRI) suggest that chronicpain alters functional connectivity between brain structures relevant tonociceptive processing. However, temporal resolution of fMRI (˜1 Hz) iswell below the frequency domain of fast neuronal ‘spiking’ activity(typically above 500 Hz) or neuronal oscillation related to cognition(between 2-250 Hz). Notably, animals used in fMRI studies are deeplyanesthetized or head-restrained, and thus, do not reflect the physiologyand different pain states of awake, freely-behaving animals.

Pain is a major symptom in many medical conditions and can significantlyinterfere with a patient's quality of life and general functioning. Thefinancial burden associated with chronic pain in the United States isestimated to be greater than $150 billion a year, due to decreasedproductivity and medical expenses. Accordingly, there exists a need inthe medical field to develop safe and effective methods of detectingpain and the use of these methods to determine efficacious therapies forthe diverse diseases and disorders associated with pain. Thus, methodscapable of detecting and monitoring pain are highly desirable.

SUMMARY OF THE INVENTION

Disclosed are methods to detect and treat pain in subjects (such as amammal (e.g., a human)) using brain activity, e.g., as determined byelectroencephalography (EEG). Additionally, methods of screening for atherapeutic agent that treats or prevents pain in a subject (e.g., anon-human mammal) are disclosed. The invention also features methods oftreating or reducing pain in a subject (e.g., a human) by stimulatingthalamic reticular nucleus (TRN) in the subject (e.g., a non-humanmammal or a human), such as with electrical stimulation, optogeneticstimulation (e.g., using a laser-emitting optic fiber adapted forimplantation in the brain of the subject), a therapeutic agent, thermalstimulation, or ultrasound stimulation. Accordingly, the invention caninclude a closed-loop system featuring, e.g., a therapeutic agent orneuromodulatory device.

A first aspect of the invention features a method for detecting pain ina subject (such as a mammal (e.g., a human)). The method includes (a)recording waveforms in brain tissue of the subject by EEG; (b) applyingfast Fourier transfer (FFT) to convert the waveforms from the timedomain to the frequency domain, thereby producing power spectral density(PSD); and (c) determining power amplitude from the PSD, in which anincrease in the power amplitude from baseline serves as an indicator ofpain. In some embodiments, the pain is selected from the groupconsisting of acute pain, inflammatory pain, and neuropathic pain.Preferably, the method further includes determining connectivity betweenbrain regions, such as the coherence of brain regions from PSD,cross-frequency coupling, or Granger causality analyses.

For example, the method can further include the step of (d) determiningcoherence of brain regions from the FFT, in which an increase in thecoherence of brain regions (e.g., the primary somatosensory cortex andprefrontal cortex) serves as an indicator of pain. In particular, thecoherence of brain regions is determined from the difference incoherence at individual frequency units or frequency bands (e.g., about3 Hz to about 30 Hz). For example, an increase in the coherence of brainregions indicates a transition from acute pain to chronic pain.

In some embodiments, the method can further include the step ofadministering a therapeutic agent to the subject (such as a mammal(e.g., a human)), e.g., to determine an effective amount of thetherapeutic agent for the treatment or prevention of pain. Inparticular, there can be a decrease in the power amplitude afteradministering the therapeutic agent relative to baseline. Additionally,there can be a decrease in the coherence of brain regions afteradministering the therapeutic agent relative to baseline. Thedetermining can also include repeating steps (a)-(d) of the method afteradministration of the therapeutic agent.

In some embodiments, the method can be performed on a second subject,e.g., in which the power amplitude from the PSD of the subject iscompared to the second subject. Moreover, the method can be performed onthe subject one or more times. The method can also be performed on asubject under anesthesia or during surgery.

The method of the first aspect can further include stimulating thalamicreticular nucleus (TRN) in the subject (e.g., a non-human mammal or ahuman), such as with electrical stimulation, optogenetic stimulation(e.g., using a laser-emitting optic fiber adapted for implantation inthe brain of the subject), a therapeutic agent, thermal stimulation, orultrasound stimulation. For example, a therapeutic agent can act onGABAergic neurons, such as therapeutic agents that target GABA receptors(e.g., barbiturates, bamaluzole, gabamide, y-Amino-o-hydroxybutyric acid(GABOB), gaboxadol, ibotenic acid, isoguvacine, isonipecotic acid,muscimol, phenibut, picamilon, progabide, quisqualamine, SL 75102, orthiomuscimol) or GABA transmitter uptake/trafficking (e.g., CI-966,deramciclane (EGIS-3886), gabaculine, guvacine (C10149), nipecotic acid,NNC 05-2090, NNC-711, SKF-89976A, SNAP-5114, tiagabine, or hyperforin).

In particular, there is a decrease in pain after stimulation of the TRNin the subject. The decrease in pain can be determined by repeatingsteps (a)-(c) of the method of the first aspect, e.g., in which adecrease in a theta frequency band from baseline indicates a reductionin pain of the subject. In particular, the TRN stimulation is at afrequency sufficient to treat or reduce pain, such as about 0.2 Hz toabout 60 Hz (e.g., about 0.2 Hz, about 0.5 Hz, about 1 Hz, about 5 Hz,about 10 Hz, about 15 Hz, about 20 Hz, about 25 Hz, about 30 Hz, about35 Hz, about 40 Hz, about 45 Hz, about 50 Hz, about 55 Hz, or about 60Hz).

A second aspect of the invention features a method of treating orreducing pain in a subject (such as a mammal (e.g., a human)) by (a)recording waveforms in brain tissue of the subject by EEG; (b) applyingFFT to convert the waveforms from the time domain to the frequencydomain, thereby producing PSD; (c) determining power amplitude from thePSD; and (d) administering a therapeutic agent to the subject if thereis an increase in the power amplitude from baseline. In someembodiments, the pain is selected from the group consisting of acutepain, inflammatory pain, and neuropathic pain. Preferably, the methodfurther includes determining connectivity between brain regions, such asthe coherence of brain regions from the FFT, cross-frequency coupling,or Granger causality analyses.

For example, the method can further include the step of (d) determiningcoherence of brain regions (e.g., the primary somatosensory cortex andprefrontal cortex) from the PSD, in which an increase in the coherenceof the brain regions serves as an indicator of pain. In someembodiments, the coherence of brain regions is determined from thedifference in coherence at individual frequency units or frequency bands(e.g., about 3 Hz to about 30 Hz). For example, an increase in thecoherence of brain regions indicates a transition from acute pain tochronic pain.

The method can further include the step of determining an effectiveamount of the therapeutic agent for the treatment or prevention of painin the subject (such as a mammal (e.g., a human)). For instance, if thetherapeutic agent is effective, there can be a decrease in the poweramplitude or coherence of brain regions after administering thetherapeutic agent relative to baseline. The determining can furtherinclude repeating steps (a)-(d) of the method after administration ofthe therapeutic agent.

The method can also include administering one or more additionaltherapeutic agents to the subject. Furthermore, the determining can beperformed, e.g., one or more times an hour, one or more times a day, orone or more times a month.

A third aspect of the invention features a method of screening for atherapeutic agent that treats or prevents pain in a subject (e.g., anon-human mammal). This method includes the steps of: (a) administeringan agent to the subject that results in behavior associated with pain(e.g., hindpaw licking and flinching); (b) recording waveforms in braintissue of the subject by EEG; (c) applying FFT to convert the waveformsfrom the time domain to the frequency domain, thereby producing PSD; (d)determining power amplitude from the PSD; (e) administering a testtherapeutic agent to the subject; and (f) repeating steps (b)-(d), inwhich a decrease in the power amplitude relative to baseline indicatesthat the test therapeutic agent treats or prevents pain in the subject.In some embodiments, the pain is selected from the group consisting ofacute pain, inflammatory pain, and neuropathic pain. Preferably, themethod further includes determining connectivity between brain regions,such as the coherence of brain regions from FFT, cross-frequencycoupling, or Granger causality analyses.

For example, the method can further include the step of determiningcoherence of brain regions (e.g., the primary somatosensory cortex andprefrontal cortex) from the PSD of the subject (e.g., a non-humanmammal). In one embodiment, the coherence of brain regions (e.g., theprimary somatosensory cortex and prefrontal cortex) is determined fromthe difference in coherence at individual frequency units or frequencybands (e.g., about 3 Hz to about 30 Hz). For example, a decrease incoherence of brain regions relative to baseline indicates that the testtherapeutic agent treats or prevents pain in the subject (e.g., anon-human mammal).

A fourth aspect of the invention features a method of treating orreducing pain in a subject (e.g., a non-human mammal or a human) thatincludes stimulating TRN in the subject with electrical current or usinga laser-emitting optic fiber adapted for implantation in the brain ofthe subject. The method can also include TRN stimulation using, e.g., atherapeutic agent, thermal stimulation, or ultrasound stimulation, totreat or reduce pain the subject. For example, a therapeutic agent canact on GABAergic neurons, such as therapeutic agents that target GABAreceptors (e.g., barbiturates, bamaluzole, gabamide,y-Amino-o-hydroxybutyric acid (GABOB), gaboxadol, ibotenic acid,isoguvacine, isonipecotic acid, muscimol, phenibut, picamilon,progabide, quisqualamine, SL 75102, or thiomuscimol) or GABA transmitteruptake/trafficking (e.g., CI-966, deramciclane (EGIS-3886), gabaculine,guvacine (C10149), nipecotic acid, NNC 05-2090, NNC-711, SKF-89976A,SNAP-5114, tiagabine, or hyperforin).

The TRN stimulation is at a frequency sufficient to treat or reduce pain(e.g., acute pain, inflammatory pain, or neuropathic pain), such asabout 0.2 Hz to about 60 Hz (e.g., about 0.2 Hz, about 0.5 Hz, about 1Hz, about 5 Hz, about 10 Hz, about 15 Hz, about 20 Hz, about 25 Hz,about 30 Hz, about 35 Hz, about 40 Hz, about 45 Hz, about 50 Hz, about55 Hz, or about 60 Hz). The method can further include determining atheta frequency band in brain tissue of the subject after the TRNstimulation, such that a decrease in the theta frequency band frombaseline indicates a reduction in pain of the subject. For example, themethod can further include: (a) recording waveforms in brain tissue ofthe subject by EEG; (b) applying fast FFT to convert the waveforms fromthe time domain to the frequency domain, thereby producing PSD; and (c)determining a theta frequency band from the PSD, such that a decrease inthe theta frequency band from baseline indicates a reduction in pain ofthe subject.

In any of the above aspects, the waveforms can be recorded with one ormore sensors (e.g., one or more electrodes) positioned on the skull ofthe subject. The waveforms can also be recorded with one or more sensors(e.g., one or more electrodes) attached to the scalp of the subject.

In any of the above aspects, the waveforms can be recorded at samplefrequencies of about 2 Hz to about 35,000 Hz (e.g., sample frequenciesof about 10 Hz to about 300 Hz). Additionally, brain activity can berecorded, e.g., by magnetoencephalography (MEG,) functional magneticresonance imaging (fMRI), or positron emission tomography (PET).

Definitions

As used herein, “a” or “an” means “at least one” or “one or more” unlessotherwise indicated. In addition, the singular forms “a,” “an,” and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to a composition containing “atherapeutic agent” includes a mixture of two or more therapeutic agents.

As used herein, “about” refers to an amount±10 of the recited value.

As used herein, “acute pain” refers to a type of pain that typicallylasts less than three to six months and/or pain that is directly relatedto soft tissue damage. Acute pain may follow non-neural tissue injury,for example, tissue damage from surgery or inflammation. Acute pain isof short duration and gradually resolves as the injured tissues heal.

As used herein, “chronic pain” refers to a type of pain that lastslonger than three to six months and/or pain that extends beyond theexpected period of tissue healing. Chronic pain may originate with aninitial trauma/injury or infection, or may be an ongoing cause of painassociated with neuropathic pain (e.g., diabetic peripheral neuropathy,post-herpetic neuralgia, trigeminal neuralgia, phantom limb pain, carpaltunnel syndrome, sciatica, pudendal neuralgia, complex regional painsyndrome, sensory polyneuropathies, mono-neuropathies, or central painsyndrome), headaches, joint pain, backaches, sinus pain, muscle pain,nerve pain, and pain affecting specific parts of the body, such asshoulders, pelvis, and neck. Chronic pain may also be associated withlower back pain, arthritis, multiple sclerosis, fibromyalgia, shingles,nerve damage, or cancer.

As used herein, “coherence” refers to the magnitude squared coherence asa measure of power transfer between stochastic systems. The output ofthe function yields coherence values between 0 and 1, with a value of 1signifying 100% perfectly matching amplitude difference between twowaveforms at the observed frequency. For example, the coherence of brainregions is determined from the difference in coherence at individualfrequency units or frequency bands (e.g., about 3 Hz to about 30 Hz).

As used interchangeably herein, the terms “decrease” and “reduce” referto the ability to cause an overall decrease preferably of 20% orgreater, more preferably of 50% or greater, and most preferably of 75%,85%, 90%, 95%, or greater. Decrease or reduce may refer to, e.g., thesymptoms of the disease, disorder, or pain in general or thedetermination of waveforms as recorded by the methods disclosed herein.

As used herein, the terms “electroencephalography” and “EEG” refer to anelectrophysiological monitoring method to record electrical activity inbrain tissue of a subject using one or more sensors attached to thescalp of a subject or with implantable sensors.

As used herein, “electrode” refers to an electric conductor throughwhich an electric current enters or leaves an electrolytic cell or othermedium. It further refers to the geometric configuration of discretetype electrical conductive elements capable of causing anelectromagnetic field when a current and voltage is applied. Theelectrode can be of any shape, and can be symmetrically orasymmetrically configured. Size and shape depend on the specificrequirements of the application.

As used herein, the phrase “fast Fourier transfer” or “FFT” is analgorithm used to convert waveforms from the time domain to thefrequency domain. FFT may be implemented using a computing programincluding a computing language, e.g., MATLAB® (MathWorks), and/or acomputing language, e.g., C, C++, Java, Fortran, or Python.

The abbreviation “fMRI,” as used herein, refers to functional magneticresonance imaging.

As used herein, the phrase “inflammatory pain” refers to a form of painthat is caused by tissue injury or inflammation (e.g., in postoperativepain or rheumatoid arthritis).

The abbreviation “MEG,” as used herein, refers tomagnetoencephalography.

As used herein, the term “naïve” refers to the state of a subject, suchas a non-human mammal, prior to induction of a pain model, as describedherein.

As used herein, the term “neuropathic pain” refers to pain caused bydamage or disease affecting the somatosensory nervous system. Forexample, neuropathic pain includes, but is not limited to, diabeticperipheral neuropathy, post-herpetic neuralgia, trigeminal neuralgia,phantom limb pain, carpal tunnel syndrome, sciatica, pudendal neuralgia,complex regional pain syndrome, sensory polyneuropathies,mono-neuropathies, or central pain syndrome, headaches, joint pain,backaches, sinus pain, muscle pain, nerve pain, and pain affectingspecific parts of the body, such as shoulders, pelvis, and neck, and/orpain that is associated with lower back pain, arthritis, headache,multiple sclerosis, fibromyalgia, shingles, nerve damage, or cancer.

The abbreviation “PET,” as used herein, refers to positron emissiontomography.

As used herein, “power spectral density” or “PSD” refers to thenumerical or visual representation (e.g., histogram) of the distributionof the power amplitude of a waveform as a function of frequency.Specific frequency bands may be evaluated using PSD, which include, butare not limited to, theta (e.g., 4-8 Hz), alpha (e.g., 8-12 Hz), beta(e.g., 12-25 Hz), and gamma (e.g., 25-100 Hz) frequency bands. Analysisof PSD outside of standard frequency bands (e.g. 6-15 Hz, 100-3000 Hz))may also be evaluated using the methods described herein.

As used herein, “prevention” refers to a prophylactic treatment given toa subject who has or will have a disease, a disorder, a condition, orone or more symptoms associated with a disease, a disorder, or acondition.

As used herein, “therapeutic agent” refers to any agent that produces ahealing, curative, stabilizing, or ameliorative effect. An “agent” mayalso be used, for example, to stimulate or cause a response in thesubject, such as behavior in response to pain, e.g., hindpaw licking andflinching, in a non-human subject. In particular, a therapeutic agentmay be included in a closed-loop system. For example, a therapeuticagent can act on GABAergic neurons to stimulate the thalamic reticularnucleus (TRN) in a subject, such as therapeutic agents that target GABAreceptors (e.g., barbiturates, bamaluzole, gabamide,y-Amino-o-hydroxybutyric acid (GABOB), gaboxadol, ibotenic acid,isoguvacine, isonipecotic acid, muscimol, phenibut, picamilon,progabide, quisqualamine, SL 75102, or thiomuscimol) or GABA transmitteruptake/trafficking (e.g., CI-966, deramciclane (EGIS-3886), gabaculine,guvacine (C10149), nipecotic acid, NNC 05-2090, NNC-711, SKF-89976A,SNAP-5114, tiagabine, or hyperforin).

As used herein, “treating” refers to administering a pharmaceuticalcomposition for prophylactic and/or therapeutic purposes. To “reduce thelikelihood” refers to prophyactic treatment of a patient who is not yetill, but who is susceptible to, or otherwise at risk of, a particulardisease or condition (e.g., the conditions described herein, such aspain (e.g., acute pain, inflammatory pain, or neuropathic pain). To“treat disease” or use for “therapeutic treatment” refers toadministering treatment to a patient already suffering from a disease toameliorate the disease and improve the patient's condition. The term“treating” also includes treating a patient to delay progression of adisease or its symptoms. Beneficial or desired results can include, butare not limited to, alleviation, amelioration, or prevention of pain, acondition associated with pain, or one or more symptoms associated withpain.

As used interchangeably herein, the terms “subject” and “patient” referto any animal (e.g., a mammal, e.g., a human). A subject to be treatedor tested for responsiveness to a therapy according to the methodsdescribed herein can be one who has been diagnosed with pain.

As used herein, the phrase “waveform” refers to an extracellular localfield potential measurement that represents the aggregate activity of apopulation of neurons. Measurements of waveforms may be used todetermine neural activity in the central nervous system, e.g., the brainand spinal cord, or in peripheral nervous system.

The recitation herein of numerical ranges by endpoints is intended toinclude all numbers subsumed within that range (e.g., a recitation of 1to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

Other features and advantages of the invention will be apparent from thefollowing Detailed Description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. A-1C are an image and a series of graphs showing electrodeplacement and representative waveforms recorded usingelectroencephalography (EEG). Screw electrodes were placedstereotaxically over the primary somatosensory cortex (S1), specificallythe ipsilateral S1(iS1) and contralateral S1 (cS1), and midlineprefrontal cortex (PFC), according to Bregma coordinates (FIG. 1A). Fora naïve rat, representative EEG cS1 waveforms down-sampled to 250 Hz andband-passed between 3-30 Hz, a spectrogram of the same EEG waveform, andthe corresponding power spectral density (PSD) are shown (FIG. 1B). Fora rat at day 7 (d7) after chronic constriction injury (CCI),representative EEG cS1 waveforms down-sampled to 250 Hz and band-passedbetween 3-30 Hz, a spectrogram of the same EEG waveform, and thecorresponding PSD of are shown (FIG. 1C).

FIGS. 2A-2C are a series of graphs showing EEG power spectra recordedfor PFC (FIG. 2A), cS1 (FIG. 2B), and iS1 (FIG. 2C) at 30 minutes aftercapsaicin, day 2 (d2) after Complete Freund's Adjuvant (CFA), and d7after CCI (shaded areas represent standard error of the mean).

FIGS. 3A-3B are a series of graphs showing EEG mean power amplitude overtime after capsaicin, CFA, or CCI (FIG. 3A) and thermal hyperalgesia, asdetermined by paw withdrawal latency (PWL), after capsaicin, CFA, or CCI(FIG. 3B).

FIGS. 4A-4B are a series of graphs showing EEG mean power amplitude overtime after capsaicin, CFA, or CCI and capsaicin, CFA, or CCI followed bytreatment with ibuprofen, pregabalin, or mexiletine (FIG. 4A). Thermalhyperalgesia for each pain model followed by treatment with ibuprofen,pregabalin, or mexiletine was also determined using PWL (FIG. 4B).

FIGS. 5A-5C are graphs showing cortical coherence between PFC and cS1(FIG. 5A), iS1 and PFC (FIG. 5B), and iS1 and cS1 (FIG. 5C) aftercapsaicin, CFA, or CCI and capsaicin, CFA, or CCI followed by treatmentwith ibuprofen, pregabalin, or mexiletine.

FIG. 6 is a graph showing control conditions using sham pain models andvehicle drug treatments.

FIG. 7 is an image of a wireless, 16-electrode, single-use EEG systemused to study waveforms in human subjects after pain.

FIGS. 8A-8C are images of the study design (FIG. 8A) and correspondingwaveforms (FIG. 8B) and source localization (FIG. 8C) of human subjectsduring a pain state, as detected using EEG.

FIGS. 9A-9D are images of extracellular in vivo recording. Shown are theassembly of the FlexDrive stereotrode system mounted with a fiberopticferrule (FIG. 9A), isolation of two putative single-units from a300-3000 Hz band-pass local field potential (FIG. 9B),channelrhodopsin-2 expression restricted to thalamic reticular nucleus(TRN) in a transgenic mouse co-expressing the vesicular GABA transporter(VGAT; FIG. 9C), and a representative coronal section showingelectrolytic lesion (circle; arrows mark tetrode track) denoting arecording site in the ventral posterolateral (VPL) thalamus (whiteshadow in right panel; FIG. 9D).

FIGS. 10A-10D are graphs showing that TRN stimulation decreases SI powerin the theta band while increasing thalamic bursts and the withdrawalthreshold in naïve VGAT mice. A histogram of the effects of TRNstimulation at 0.5, 10, and 50 Hz on mean theta (4-8 Hz) power under1.5% isoflurane sedation is shown (n=2 mice; FIG. 10A). SI power spectraare shown, in which the right panel inset shows a significant decreasein power within the theta band (3.8-8.5 Hz) following 10 Hz TRNstimulation in awake mice (n=5 mice; FIG. 10B). TRN stimulationincreases burst firing in VPL neurons (n=17 units, 3-4 units per mouse;5 mice) and increases the threshold of mechanical withdrawal to von Freystimuli (d; n=4 mice; FIG. 10C-D).

FIGS. 11A-11E are graphs showing that TRN stimulation during acute painrescues SI theta power and reverses allodynia. SI power spectra areshown, in which the right panel inset shows increased power within thetheta band (3.8-6.2 Hz) following capsaicin compared to naïve mice,whereas TRN stimulation reverses these changes (n=5 mice; FIG. 11A).Capsaicin increases burst firing in VPL neurons, which is furtherenhanced following TRN stimulation (n=17 units, 5 mice; FIG. 11B).Withdrawal thresholds following capsaicin indicate tactile allodynia,which is reversed upon TRN stimulation, but re-emerges 5 minutesafterwards (n=7 mice, FIG. 11C). A spectrogram illustrating the temporaldynamics of SI theta in relation to bursts in the VPL under naive,capsaicin, and capsaicin plus optogenetic conditions is shown (arrowheadmarks light onset; gray line marks duration of optical stimulation; FIG.11D). Note that theta and burst epochs do not temporally coincide.Dynamic, time-lagged cross-correlation between SI theta power relativeto tonic and burst firing shows a significant negative correlationbetween theta-bursts when bursts precede theta by 120 ms (n=17 units; 5mice; FIG. 11E).

FIG. 12 is a graph showing a summary of EEG results in different painmodels. Black cells represent significant increases compared to naïverats.

DETAILED DESCRIPTION OF THE INVENTION

There is alack of reliable methods available for detecting andmonitoring pain, particularly for determining effective therapeuticagents for a variety of conditions, disorders, and diseases associatedwith pain. I have developed a method of detecting pain in a subject(such as a mammal, e.g., a human) by recording waveforms in brain tissueusing electroencephalography (EEG), applying fast Fourier transfer (FFT)to convert the waveforms from the time domain to the frequency domain,thereby producing power spectral density (PSD), and then determiningpower amplitude from the PSD. The methods disclosed herein can also beused, e.g., to treat or reduce pain in a subject, e.g., by administeringa therapeutic agent to the subject, if there is an increase in the poweramplitude from baseline. In particular, the methods are useful fordetecting and treating or reducing acute pain, inflammatory pain, andneuropathic pain. Additionally, the methods can be used to screen fortherapeutic agents that decrease power amplitude, and thus, treat orprevent pain in the subject. The invention also features methods totreat or reduce pain in a subject (such as a mammal, e.g., a human) bystimulating thalamic reticular nucleus (TRN) in the subject, such aswith electrical stimulation, optogenetic stimulation (e.g., using alaser-emitting optic fiber adapted for implantation in the brain of thesubject), a therapeutic agent, thermal stimulation, or ultrasoundstimulation. Thus, the methods can feature a closed loop systemincluding, e.g., a closed-loop system featuring, e.g., a therapeuticagent or neuromodulatory device.

Diagnostic Methods

Neuronal activity in a subject may be detected at the level of waveformsusing EEG. In particular, analysis of waveforms in brain tissue usingEEG allows for the study of multiple neuronal networks simultaneously.Waveforms may be recorded at sampling frequencies between about 2 Hz toabout 35,000 Hz. Preferably, waveforms are recorded at samplefrequencies between about 3 Hz to about 300 Hz. Waveforms may berecorded via EEG with one or more sensors (e.g., electrodes) positionedon the skull of the subject or with one or more sensors (e.g.,electrodes) attached to the scalp of the subject. Other types of sensorsinclude any sensor capable of detecting neuronal activity, e.g., calciumimaging, fMRI, MEG, MRI, and PET (acronyms defined below).

Neuronal waveforms may be detected by EEG with invasive methods (e.g.,intraoperative or implantable sensors) or non-invasive methods (such assensors, e.g., electrodes, attached to the scalp of a subject). Thesemethods can include detecting shifts in PSD using FFT analysis todetermine the occurrence or absence of new spectral peaks, shifts inpeak amplitudes or peak latency from a PSD. Methods of detectingwaveforms in brain tissue of a subject may further include the use ofmagnetoencephalography (MEG) in addition to other types of imagingtechniques and brain scans (for example, magnetic resonance imaging(MRI), functional magnetic resonance imaging (fMRI), and positronemission tomography (PET)) in combination with EEG. Such techniques maybe applied to a subject prior to, concurrently, or subsequent torecording of waveforms using EEG.

Thus, the present invention provides methods for detecting waveforms inbrain tissue of a subject (e.g., a mammal, e.g., a human) indicative ofpain using EEG. These methods feature the detection of waveforms inbrain tissue of a subject, e.g., as a biomarker for pain, such as acutepain, inflammatory pain, and neuropathic pain. The neuronal activitypatterns that make up the pain biomarker can be divided into two majorcategories: spontaneous (e.g., independent or temporally not associatedwith an overt stimulus or identifiable cause) and evoked (e.g., activitycorrelated with an overt stimulus or identifiable cause). Both forms ofpain may be detected using these methods.

The methods can also be performed one or more (e.g., two, there, four,or five) times to detect waveforms in brain tissue of a subject (e.g., amammal, e.g., a human) indicative of pain using EEG at intervals (e.g.,in seconds, minutes, or in hours), irregularly, or continuously. Inparticular, the methods using EEG are performed in intervals of seconds,such as for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25,30, 35, 40, 45, 50, 55, or 60 seconds, to detect waveforms in braintissue indicative of pain.

Pain

Pain is associated with a wide range of medical conditions. The presentinvention features methods for diagnosing and treating a subject (e.g.,a mammal, such as a human) with pain or conditions associated with pain.The methods of diagnosis and treatment are based, inter alia, on theinventor's discovery that waveforms in brain tissue of a subjectdetected by EEG are indicative of pain. Subjects diagnosed and treatedusing the methods can include subjects with acute pain, subacute pain,or chronic pain (e.g., pain that lasts longer than three to six monthsor pain that extends beyond the expected period of healing); orconditions associated with pain (e.g., post-herpetic neuralgia,trigeminal neuralgia, phantom limb pain, carpal tunnel syndrome,sciatica, pudendal neuralgia, complex regional pain syndrome, or centralpain syndrome, headaches, in particular, migraine, joint pain,backaches, sinus pain, muscle pain, nerve pain, and pain affectingspecific parts of the body, such as shoulders, pelvis, and neck, and/orpain that is associated with lower back pain, arthritis, headache,fibromyalgia, shingles, or nerve damage).

Methods described herein may be useful for the diagnosis, treatment,reduction, or prevention of various forms of pain, whether acute orchronic. Exemplary conditions that may be associated with pain include,for example, soft tissue, joint, and bone inflammation and/or damage(e.g., acute trauma, osteoarthritis, or rheumatoid arthritis),myofascial pain syndromes (fibromyalgia), headaches (including clusterheadache, migraine, and tension type headache), myocardial infarction,angina, ischemic cardiovascular disease, post-stroke pain, sickle cellanemia, peripheral vascular occlusive disease, cancer, inflammatoryconditions of the skin or joints, diabetic neuropathy, and acute tissuedamage from surgery or traumatic injury (e.g., burns, lacerations, orfractures).

For example, the present invention provides methods for detecting andtreating inflammatory pain. Inflammatory pain is a form of pain causedby tissue injury or inflammation (e.g., in postoperative pain orrheumatoid arthritis). Following a peripheral nerve injury, symptoms aretypically experienced in a chronic fashion, distal to the site of injuryand are characterized by hyperesthesia (enhanced sensitivity to anatural stimulus), hyperalgesia (abnormal sensitivity to a noxiousstimulus), allodynia (widespread tenderness associated withhypersensitivity to normally innocuous tactile stimuli), and/orspontaneous burning or shooting lancinating pain. In inflammatory pain,symptoms are apparent, at least initially, at the site of injury orinflamed tissues and typically accompany arthritis-associated pain,musculo-skeletal pain, and postoperative pain. The different types ofpain may coexist or pain may be transformed from inflammatory toneuropathic during the natural course of the disease, as inpost-herpetic neuralgia.

Additionally, the present invention provides methods for detecting andtreating neuropathic pain. Neuropathic pain can take a variety of formsdepending on its origin and can be characterized as acute, subacute, orchronic depending on the duration. Acute pain can last anywhere from acouple hours to less than 30 days. Subacute pain can last from one tosix months and chronic pain is characterized as pain that lasts longerthan three to six months or pain that extend beyond the expected periodof healing. In neuropathic pain, the pain may be described as beingperipheral neuropathic if the initiating injury occurs as a result of acomplete or partial transection of a nerve or trauma to a nerve plexus.Peripheral neuropathy can result from traumatic injuries, infections,metabolic disorders, diabetes, and/or exposure to toxins. Alternatively,neuropathic pain is described as being central neuropathic following alesion to the central nervous system, such as a spinal cord injury or acerebrovascular accident. The methods of the invention includeadministration of the compositions described herein to treat neuropathicpain. Types of neuropathic pain include but are not limited to: diabeticperipheral neuropathy, post-herpetic neuralgia, trigeminal neuralgia,phantom limb pain, carpal tunnel syndrome, sciatica, pudendal neuralgia,complex regional pain syndrome, sensory polyneuropathies,mono-neuropathies, and central pain syndrome.

The present invention may also be useful for the diagnosis, treatment,reduction, or prevention of musculo-skeletal pain (after trauma,infections, and exercise), pain caused by spinal cord injury, tumors,compression, inflammation, dental pain, episiotomy pain, deep andvisceral pain (e.g., heart pain, bladder pain, or pelvic organ pain),muscle pain, eye pain, orofacial pain (e.g., odontalgia, trigeminalneuralgia, glossopharyngeal neuralgia), abdominal pain, gynecologicalpain (e.g., dysmenorrhea and labor pain), pain associated with nerve androot damage due to trauma, compression, inflammation, toxic chemicals,hereditary conditions, central nervous system pain, such as pain due tospinal cord or brain stem damage, cerebrovascular accidents, tumors,infections, demyelinating diseases including multiple sclerosis, lowback pain, sciatica, and post-operative pain.

Methods of Treatment

The present invention provides methods of treating or reducing pain in asubject (e.g., a mammal, such as a human) by recording waveforms inbrain tissue of the subject using EGG, applying FFT to convert waveformsfrom the time domain to the frequency domain, thereby producing PSD,determining power amplitude from the PSD, and administering atherapeutic agent to the subject, if there is an increase in the poweramplitude from baseline. Additionally, waveforms recorded in braintissue of a subject by EEG can be used to determine coherence of brainregions, in which an increase in the coherence of brain regions (e.g.,the PFC and S1) is indicative of, e.g., a transition from acute pain tochronic pain. Accordingly, a therapeutic agent can be administered afterdetermining an increase in brain region coherence. Thus, the methodsresult in a reduction in the likelihood of pain or prevention of pain.

The methods of the present invention for treating or reducing pain in asubject may be performed on the subject within 24 hours (e.g., within 20hours, 16 hours, 12 hours, 8 hours, 4 hours, 3 hours, 2 hours, or 1hour) of an initial presentation of the subject to a medicalprofessional. The method may also be performed at least 24 hours (e.g.,at least 48 hours, 3 days, 4 days, 5 days, 6 days, or one week) after aninitial presentation of the subject to a medical professional. Themethod may be performed on a subject previously admitted to a medicalfacility for a disease or disorder. The method may also be performed oneor more (e.g., two, there, four, or five) times for treating a subjectat intervals (e.g., hourly, daily, weekly, or monthly) or irregularly.

Upon assessing that there is an increase in the power amplitude frombaseline, a therapeutic agent may be administered to the subject one ormultiple times daily (e.g., two times, three times, up to four times aday), weekly (or at some other multiple day interval), or on anintermittent schedule, with that cycle repeated a given number of times(e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles) or indefinitely. Accordingto the methods described herein, therapeutic agents may also beadministered chronically (e.g., more than 20 days, e.g., 21 days, 30days, 60 days, 3 months, 6 months, 9 months, 1 year, 2 years, or 3years). Sensors of the present method may also be coupled to an‘effector’ (e.g. pharmacotherapy or neuromodulatory device) in anautomated closed-loop system.

The present invention also provides methods of treating or reducing pain(e.g., acute pain, inflammatory pain, or neuropathic pain) in a subject(e.g., a mammal, such as a human) by stimulating thalamic reticularnucleus (TRN) in the subject. In particular, methods of treating orreducing pain in a subject feature stimulation of TRN using, e.g.,electrical stimulation, optogenetic stimulation (e.g., using alaser-emitting optic fiber adapted for implantation in the brain of thesubject), a therapeutic agent, thermal stimulation, or ultrasoundstimulation. For example, the TRN can be stimulated at a frequency ofabout 0.2 Hz to about 100 Hz, such as about 0.2 Hz, about 0.5 Hz, about1 Hz, about 5 Hz, about 10 Hz, about 15 Hz, about 20 Hz, about 25 Hz,about 30 Hz, about 35 Hz, about 40 Hz, about 45 Hz, about 50 Hz, about55 Hz, about 60 Hz, about 65 Hz, about 70 Hz, about 75 Hz, about 80 Hz,about 85 Hz, about 90 Hz, about 95 Hz, or about 100 Hz. In particular,TRN stimulation can be intermittent or ‘burst’ stimulation, such asabout 100 Hz to about 200 Hz bursts of individual stimulation epochs.Additionally, the TRN of the subject can be stimulated with alaser-emitting optic fiber one or multiple times daily (e.g., two times,three times, up to four times a day), weekly (or at some other multipleday interval), or on an intermittent schedule, with that cycle repeateda given number of times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles), orindefinitely. For example, a therapeutic agent may also be administeredto the subject to stimulate the TRN in the subject, thereby treating orreducing pain in the subject. In particular, a therapeutic agent cantarget GABA receptors (e.g., barbiturates, bamaluzole, gabamide,y-Amino-o-hydroxybutyric acid (GABOB), gaboxadol, ibotenic acid,isoguvacine, isonipecotic acid, muscimol, phenibut, picamilon,progabide, quisqualamine, SL 75102, or thiomuscimol) or GABA transmitteruptake/trafficking (e.g., CI-966, deramciclane (EGIS-3886), gabaculine,guvacine (C10149), nipecotic acid, NNC 05-2090, NNC-711, SKF-89976A,SNAP-5114, tiagabine, or hyperforin).

The methods of the present invention for treating or reducing pain in asubject (e.g., a mammal, such as a human) featuring TRN stimulation maybe performed on the subject within 24 hours (e.g., within 20 hours, 16hours, 12 hours, 8 hours, 4 hours, 3 hours, 2 hours, or 1 hour) of aninitial presentation of the subject to a medical professional. Themethod of TRN stimulation may also be performed at least 24 hours (e.g.,at least 48 hours, 3 days, 4 days, 5 days, 6 days, or one week) after aninitial presentation of the subject to a medical professional. Themethod of TRN stimulation may be performed on a subject previouslyadmitted to a medical facility for a disease or disorder associated withpain (e.g., acute pain, inflammatory pain, or neuropathic pain). Themethod of TRN stimulation may also be performed one or more (e.g., two,there, four, or five) times for treating a subject at intervals (e.g.,hourly, daily, weekly, or monthly) or irregularly. Additionally, TRNstimulation may be performed on a subject having pain as determined by,e.g., recording waveforms in brain tissue of the subject by EEG;applying FFT to convert the waveforms from the time domain to thefrequency domain, thereby producing PSD; and determining power amplitudefrom the PSD, in which an increase in the power amplitude from baselineserves as an indicator of pain. The subject may also have not previouslyreceived treatment for pain prior to the methods.

Dosing of Therapeutic Agents

Methods of the present invention may be used to determine the effectiveamount of the therapeutic agent (e.g., dosage or titration) to treat orprevent the likelihood of pain in a subject (such as a mammal, e.g., ahuman). In particular, an effective amount of the therapeutic agentresults in, e.g., an amelioration or stabilization of pain in thesubject, such that there is a decrease in the power amplitude of the PSDrelative to baseline.

The recording, applying, and determining steps of the method may berepeated after administration of the therapeutic agent in order todetermine an effective amount of the agent. These steps may be repeatedone or more times an hour (e.g., within 1 minute, 5 minutes, 10 minutes,15 minutes, 30 minutes, 45 minutes), day (e.g., within 12 hours, 8hours, 4, hours, 2 hours, 1 hour), or month (e.g., at least 48 hours, 3days, 4 days, 5 days, 6 days, or one week). Suitable therapeutic agentsalso include combinations thereof, such that one or more (e.g., two,three, four, or five or more) additional therapeutic agents isadministered to the subject. When co-administered, the two therapeuticagents are desirably administered within 24 hours of each other (e.g.,within 12 hours, 8 hours, 4, hours, 2 hours, 1 hour, 30 minutes, 15minutes, or substantially simultaneously).

Actual dosage levels of the active ingredients in the therapeutic agentsadministered according to the present invention may be varied so as toobtain an amount of the active ingredient which is effective to achievethe desired response of treating or reducing the likelihood of pain in asubject, without undesirable side effects or being toxic to the subject(such as a mammal, e.g., a human). According to the methods of thepresent invention, the selected dosage level can be determined byrecording waveforms in brain tissue of the subject by EEG. For instance,after administering an agent to the subject that results in behaviorassociated with pain, assessment of a decrease in the power amplituderelative to baseline indicates that the test therapeutic agent treats orprevents pain in the subject and can be used to select the appropriatedosage of the test therapeutic agent. Additionally, side effectsassociated with analgesics (e.g., drowsiness with gabapentanoids or anincrease in coherence values with mexiletine) can be determined with themethods.

The selected dosage level will also depend upon a variety ofpharmacokinetic factors including the activity of the therapeuticagents, the route of administration, the time of administration, therate of absorption of the particular agent being employed, the durationof the treatment, other drugs, substances, and/or materials used incombination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thesubject being treated, and like factors well known in the medical arts.It is to be understood that, for any particular subject, specific dosageregimes should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compound. For example, the dosage of atherapeutic agent can be increased if the lower dose does not providesufficient activity to decrease power amplitude relative to baseline asassessed by the methods described herein. Conversely, the dosage of atherapeutic agent may be maintained or decreased if there is anappreciable decrease in power amplitude relative to baseline.

Therapeutic agents can include, pharmacological, non-pharmacological,and neuromodulatory agents (e.g. deep brain stimulation, spinal cordstimulation, transcranial current stimulation, transcranial magneticstimulation, and ultrasound stimulation). In particular, therapeuticagents useful in the methods include non-steroidal anti-inflammatorydrug (NSAIDs). Exemplary NSAIDs include, without limitation, ibuprofen,aceclofenac, acemetacin, acetaminophen, aloxiprin, aspirin, benorilate,bromfenac, celecoxib, deracoxib, diclofenac, diflunisal, ethenzamide,etodolac, etofenamate, etoricoxib, fenbufen, fenoprofen, flufenamicacid, flurbiprofen, lonazolac, lornoxicam, indomethacin, isoxicam,kebuzone, ketoprofen, ketorolac, licofelone, loxoprofen, lumiracoxib,meclofenamic acid, mefenamic acid, meloxicam, metamizol, mofebutazone,naproxen, nabumetone, niflumic acid, nimesulide, oxaprozin,oxyphenbutazone, parecoxib, phenidone, phenybutazone, piroxicam,propacetamol, propyphenazone, rofecoxib, salicyamide, sulfinpyrazone,sulindac, suprofen, tiaprofenic acid, tenoxicam, or tolmetin.Therapeutic agents useful in the methods can also includeanticonvulsants, such as pregabalin, carbamazepine, flupirtine,gabapentin, lamotrigine, oxcarbazepine, phenytoin, retigabine,topiramate, or valproate. Additionally, useful therapeutic agents of themethods include antiarrhythmic agents, such as mexiletine, lidocaine, ortocainide.

Methods of Screening Therapeutic Agents

The present invention features methods of screening for a therapeuticagent using a non-human animal subject (e.g., mammal) that includeadministering an agent to the subject that results in behaviorassociated with pain (e.g., hindpaw licking and flinching); recordingwaveforms in brain tissue of the subject by EEG, applying FFT to convertthe waveforms from the time domain to the frequency domain, therebyproducing PSD; determining power amplitude from the PSD; administering atest therapeutic agent to the subject; and repeating the priorrecording, applying, and determining steps. In particular, a decrease inthe power amplitude relative to baseline indicates that the testtherapeutic agent treats or prevents pain in the subject.

Test therapeutic agents of the present invention may be screened from aplurality of chemical entities. The steps of screening for a therapeuticagent may be repeated with one or more compounds, e.g., with a libraryof compounds. For instance, the invention may feature a librarycomprising compounds or complexes that may treat or reduce thelikelihood of pain the subject. Screening of multiple compounds can becarried out simultaneously or concurrently; or can be carried outsimultaneously with some compounds and then concurrently with others.Therapeutic agents may include pharmacological, non-pharmacological, andneuromodulatory agents, as described herein.

Clinical Applications

In addition to using the methods of the present invention for detectingpain in a subject (such as a mammal, e.g., a human) and/or treating orreducing the likelihood of pain, the present methods may be used duringinvasive or surgical procedures (e.g., intraoperative, awake lightsedation, or unconscious deep anesthesia), in particular if anestheticsor sedatives are contraindicated. Furthermore, the diagnostic methods ofthe present invention are useful for subjects or patients that arenon-cooperative, in a non-communicating vegetative state, cognitivelyimpaired, facing language barrier, or where verbal reporting isunreliable (e.g., in pediatric neonate subjects).

Methods of the present invention also provide for safe, effective, andlong-term treatment strategies for pain using, e.g., a neuromodulatorysystem for the relief of chronic pain. The methods may also includeproviding therapeutic neurostimulation to the brain of the patient,e.g., at predefined times, frequencies, voltages, periodicities, andcurrents. For instance, these methods can involve electrodes implantedinto a subjects brain, e.g., a deep brain stimulation system, electrodeson the scalp, e.g., a transcranial direct current stimulation system,and/or the use of magnetic stimulation, e.g., a transcranial magneticstimulation system. The neurostimulation can be provided in response todetecting an increase in EEG power amplitude indicative of pain or on aperiodic basis (e.g., every 1-2 hours). Methods of the present inventioncan also include the use of a transdermal patch placed on the skin fordrug delivery or an intrathecal drug delivery pump for direct deliveryof medication.

The following examples are intended to illustrate, rather than limit,the disclosure. These studies feature the use of EEG recording methodsin awake, freely-behaving rats to demonstrate that pain modulatesneuronal oscillations in clinically relevant models and that effectiveanalgesic drugs reverse this modulation. These results suggest thatrecording waveforms in brain tissue of subjects using EEG can be used topredict spontaneous nociceptive states in rodents and that waveformsassociated with pain can be used for diagnostic and therapeuticpurposes.

Example 1. Electrophysiological Measurements Using Clinically TetheredElectrodes

Experiments were performed on male Sprague-Dawley rats (n=43 rats,weight of 200 to 300 g). Animals were housed under a 12-hour light/darkcycle in a temperature- and humidity-controlled environment. Under deepanesthesia (isoflurane, 3.5%), the head was fixed in a stereotaxicapparatus. A small skin incision was used to expose the skull. Twostainless steel screw electrodes (0-80 ga, ⅛-inch, and impedance of 0.6Ohm; Component Supply Company, Fort Meade, Fla.) were placed over theintact skull corresponding to the primary somatosensory cortex (S1)hindlimb area bilaterally without craniotomy (Bregma −2, 2 mm lateral)and a third screw was placed over the area corresponding to theprefrontal cortex (PFC; Bregma +3.5 mm, midline). Minimal craniotomieswere used to place three stabilization screws (corresponding to Bregma+1.4, 2 mm bilaterally and Bregma −4.8 mm, midline) to anchor all EEGelectrodes chronically using dental acrylic. EEG screws were threadedwith a silver wire and attached to a female miniature pin connector (A&MSystems, Sequim, Wash.). Signal reference was provided by a silver wirepermanently threaded to skin at the back of the neck.

EEG recordings began five to seven days after implantation ofcontralateral S1 (cS1), ipsilateral S1(iS1), and PFC electrodes, asdescribed above (FIG. 1A). EEG waveforms were amplified (DAM80, WorldPrecision Instruments, Sarasota, Fla.), led to a processing system(micro1401mkII, Cambridge Electronic Design (CED), Cambridge, UK), andanalyzed off-line using Spike 2 (CED) or MATLAB® (Mathworks, R2012b,Natick, Mass.). Prior to EEG recording, pin connectors from eachelectrode were tethered to pre-amplifier headstages leading to amultichannel amplifier (iso-DAMA, WPI Inc., Sarasota, Fla.).Amplification for each channel was set at ×1000. This system allowedfree movement of tethered rats with no head restraint, while recordingEEG signals simultaneously from all electrodes (cS1, iS1, and PFC). Ratswere allowed to freely navigate individually in Plexiglas chambers. Therat's behavior was visually monitored, noting periods of rest. Each EEGrecording session was approximately five minutes per animal,irrespective of the pain model. Of that 5 minute interval, 15 secondsegments were selected randomly during the rest state with one 15 secondsegment selected per condition and per animal. After 15 minutes ofacclimation, EEG waveforms were sampled at 25 kHz and down-sampledoffline to 250 Hz.

Only data during awake, resting periods (defined as alertness with nolocomotor behavior) were further analyzed. Potentials generated due tovigorous myogenic activity, such as scratching, were excluded fromanalysis. These artifacts were identified by monitoring the animal'sbehavior, voltage amplitude, and spectral frequency (e.g., greater than30 Hz). Study exclusion criteria included signs of skin infection due tosurgical complications from the EEG implant or low signal-to-noise ratioindicating faulty electrophysiological signal transmission. No rat wasexcluded from the capsaicin or Complete Freund's Adjuvant (CFA) groups.Three rats were excluded from the chronic constriction injury (CCI)treatment group due to high noise in the electrophysiological signal ata later stage of CCI.

Example 2. Pain Models, Thermal Sensitivity, and Analgesic Treatment

Seven days after implantation of EEG electrodes, different pain modelswere induced. For capsaicin as a model of acute pain, capsaicin (0.1%,40 μL, Sigma-Aldrich) was intradermally injected in the left hindpawunder brief isoflurane anesthesia (1.5% for 2 minutes). A transientreceptor potential vanilloid 1 agonist, capsaicin increases neuronalfiring in nociceptors, mainly polymodal C-fibers, and is commonly usedas a model of acute nociceptive pain. Within 24 hours after capsaicininjection, nocifensive behavior indicative of spontaneous pain, such ashindpaw licking and flinching, completely subsides. Sham capsaicin ratsreceived only vehicle injections (20 μL, 7% Tween 80 in saline).

For Complete Freund's Adjuvant (CFA) as a model of inflammatory pain,CFA (100 μL, intradermal, Sigma-Aldrich) was injected in the lefthindpaw under brief isoflurane anesthesia (1.5% for 2 minutes).CFA-induced nociceptive behaviors result from the edema caused by theinflammatory response to heat-killed Mycobacterium tuberculosis in theinoculate and persists for more than 2 days after injection. Sham CFArats received vehicle injections (100 μL, incomplete Freund's adjuvantas 85% paraffin oil and 15% mannide monooleate).

For chronic constriction injury (CCI) as a model of neuropathic pain,the left sciatic nerve was exposed unilaterally after skin incision atthe mid-thigh level and blunt dissection of the biceps under deepanesthesia (isoflurane, 3.5%). Four chromic gut (4-0) ligatures weretied loosely around the nerve 1 mm apart, and the overlying muscles andskin were closed in layers with 4-0 Ethilon™ sutures. A minormodification was introduced, consisting of loose ligatures, to minimizenerve damage and deafferentation. Rats with this slightly modified CCIprocedure gradually develop typical signs of sensory hypersensitivityassociated with neuropathic pain, such as guarding the affected hindpawand thermal hypersensitivity, for more than 2 weeks after CCI. Sham CCIanimals underwent the same procedures without nerve ligation.

Thermal sensitivity of the hindpaw was assessed by measuring the latencyof the withdrawal reflex in response to a radiant heat source.Individual animals were placed in a Plexiglas box on an elevated glassplate under which a radiant heat source (4.7 amps) was applied to theplantar surface of the hindpaw after 15 minutes of acclimation. Pawwithdrawal latencies (PWL) in response to four thermal stimulations,separated by five minutes of rest, were averaged for each paw. Ratsunresponsive to radiant heat stimuli were excluded from PWL dataanalysis.

For analgesic treatment, ibuprofen was dissolved in a 5% solution of2-hydroxypropy-p-cyclodextrin (Sigma-Aldrich) formulated to deliver 30mg/kg in a volume of 3 m/kg. Pregabalin was dissolved in 5% Tween 80(Sigma-Aldrich) in saline. Mexiletine was dissolved in saline forintraperitoneal (i.p) delivery of 10 mg/kg in a volume of 3 m/kg. EEGwas performed 30 min after i.p. delivery of analgesics. Ibuprofen wasadministered concomitantly with capsaicin to allow at least 30 minutesfor the analgesic effects to manifest. Pregabalin was administered atday 2 (d2) after CFA treatment and day 14 (d14) after CCI treatment.Mexiletine was administered at day 16 (d16) after CCI treatment in thesame rats that received pregabalin to allow within group comparison ofanalgesic effects.

Example 3. Analysis of EEG Waveform Recordings

Fast Fourier transform (FFT) was used to convert EEG waveforms from thetime domain to the frequency domain, yielding power spectra. Powervalues were generated in 27 frequency bins between 3 and 30 Hz. For eachexperimental condition, 15 second continuous segments during completerest were selected for power analysis.

The magnitude squared coherence function (mscohere) in MATLAB® SignalProcessing Toolbox or the “COHER” script in Spike 2 was used as ameasure of power transfer between stochastic systems. The output of thefunction yields coherence values between 0 and 1, with a value of 1signifying perfectly matching amplitude difference between two waveformsat the observed frequency. For signals x and y, the magnitude squaredcoherence is a function of their power spectral densities P_(xx)(f) andP_(yy)(f) and their cross power spectral density P_(xy)(f):C _(xy)(f)=|P _(xy)(f)² /P _(xx)(f)P _(yy)(f)

The function parameters were defined as follows: the fast Fouriertransfer length (“nfft”) is the next power of 2 greater than the lengthof each signal, the sampling frequency (“fs”) is 250, the window length(“window”) is the periodic Hamming window to obtain 8 equal sections ofeach signal, and the number of overlapping samples (“noverlap”) is thevalue yielding 50% overlap. To minimize type I errors, coherence valueswere down-sampled from 54 to 27 frequency bins between 3 and 30 Hz.Two-way ANOVA analysis followed by Bonferroni's correction was used tocompute statistical significance. Bartlett's test was performed tocompute normal distribution and equal variance. A ‘p’ value <0.05 wasconsidered significant (denoted with * in figures). All values arereported as ±standard error of the mean.

Example 4. EEG Power, Pain, and Nociceptive Behavior

EEG recordings were performed in awake, freely-behaving rats duringrest. EEG waveforms were generally stable over time, allowing for areliable analysis of longitudinal EEG data. When 10 second interval EEGwaveforms (sampling frequency of 250 Hz) were band-pass filtered between3-30 Hz, increased voltage amplitude and oscillations were evident incorresponding spectrograms and power spectra of the cS1 of a rat atseven days following CC1 relative to a naïve rat (FIG. 1B-1C). Inparticular, the spectrogram of the naïve versus the CC1 treated ratrevealed increased low-frequency power (<10 Hz) in the cS1 at seven daysfollowing CC1.

EEG power waveforms from iS1, cS1, and PFC relevant to acute (capsaicin,n=8 rats), inflammatory (d2 after CFA, n=10 rats for PFC and cS1, n=4rats for iS1), and neuropathic pain states (d14 after CCI, n=5 rats forPFC and cS1, n=6 rats for iS1) are shown in FIG. 2. Compared to naïverats, EEG power amplitude in the 3-30 Hz range of the iS1, cS1, or PFCwas more synchronized following CFA or CCI (FIG. 2A-2C). There was noremarkable difference of EEG power spectra between iS1, cS1, or PFC,suggesting that pain is associated with widespread synchronization ofEEG. Interestingly, capsaicin, which evokes a transient and relativelyless pronounced state of nociception within 30 minutes after intradermalinjection, resulted in a modest increase in EEG power amplitude comparedto CFA and CCI, which arguably evoke a more heightened nociceptivestate. EEG power amplitude increased in the three pain models, exceptfor power recorded over iS1, which remained unchanged in rats withcapsaicin.

Mean EEG power (mV²×10⁻⁵) between 3 Hz to 30 Hz of the cS1, iS1, and PFCfollowed an ascending, linear trend during the development ofinflammatory pain due to CFA and neuropathic pain due to CCI (FIG. 3A).For cS1, EEG mean power was not changed 30 min after capsaicin(1.05±0.11, n=8 rats) compared to naïve rats (from 0.85±0.15) or 24hours after capsaicin (0.89±0.12). In contrast, CFA increased EEG meanpower from 0.54±0.10 (naïve) to 0.81±0.10 and 0.91±0.09 within one andtwo days, respectively (p<0.05, n=10 rats). CCI increased EEG mean powerfrom 0.67±0.13 (naïve) to 1.03±0.15, 1.25±0.23, and 1.39±0.21 at 7, 14,and 16 days after injury, respectively (p<0.05, n=5 rats).

For iS1, mean power increased from 0.91±0.13 (naïve) to 1.05±0.8 at 30min after capsaicin (p<0.05), and reversed 24 hours after capsaicin tonaïve levels (0.90±0.18; n=8 rats). CFA increased mean power from0.76±0.19 (naïve) to 1.13±0.20 and 1.53±0.03 within one and two days,respectively (p<0.05, n=4 rats). CCI increased mean power from 0.52±0.07(naïve) to 0.75±0.08, 1.11±0.16, and 1.22±0.10 at 7, 14, and 16 daysafter injury, respectively (p<0.05, n=6 rats).

For PFC, mean EEG power increased from 0.56±0.08 (naïve) to 0.67±0.06 at30 min after capsaicin (p<0.05), and reversed 24 hours after capsaicinto naïve levels (0.65±0.07; n=8 rats). CFA increased mean power from0.46±0.07 (naïve) to 0.72±0.08 and 0.76±0.06 within one and two days,respectively (p<0.05, n=10 rats). CCI increased mean power from0.58±0.06 (naïve) to 0.86±0.08, 1.06±0.10 and 1.08±0.16 at 7, 14, and 16days after injury, respectively (p<0.05, n=5 rats).

In summary, nociceptive states in rat models of acute, inflammatory, andneuropathic forms of pain were discovered to correlate with increasedEEG power over cS1 and PFC. Notably, EEG power in S1 ipsilateral aftercapsaicin injection was not significantly changed. These data furthersuggest that power spectra in iS1 to noxious stimuli might encodelong-lasting, but not transient forms of pain, indicating that S1 iscritical for sensory discrimination and localization of acute, noxiousstimuli on the contralateral side of the body. Notably, intradermalcapsaicin injection elicits pain that has maximal intensity immediatelyupon injection with rapid decay within 5 minutes. Secondary hyperalgesiaoccurs at a later time point starting at 10 minutes after injection andpersists at least 20 minutes thereafter. In the present study, capsaicinwas injected under brief (2-3 minute) isoflurane sedation and collectionof EEG data began 30 minutes after injection. Accordingly, the presentEEG data correspond to a time point of secondary, not primary,hyperalgesia. Thus, long-term pain leads to widespread increases in EEGpower according to an anatomical representation that does not strictlyoverlap with the cortical projection map of the spinothalamic system.

Example 5. Relationship of EEG Power and Thermal Hyperalgesia

The relationship between EEG power and thermal hyperalgesia, awidely-used correlate of pain-induced behavioral hypersensitivity, wasthen determined. Thermal hyperalgesia developed reliably in all painmodels as determined from paw withdrawal latencies (PWL; FIG. 3B). PWLdecreased from 9.43±0.22 seconds to 6.00±0.24 seconds at 30 min aftercapsaicin (p<0.05), and reversed to 9.15±0.26 seconds at 24 hour aftercapsaicin (n=8 rats). CFA decreased PWL from 9.67±0.29 seconds (naïve)to 5.73±0.36 seconds and 6.10±0.33 seconds within one and two days,respectively (p<0.05, n=10 rats). CCI decreased PWL from 8.71±0.21seconds (naïve) to 7.03±0.36, 7.00±0.24, and 7.26±0.45 seconds at 7, 14,and 16 days after injury, respectively (p<0.05, n=7 rats). Notably, themodulation of mean power versus PWL was not identical. For example, inrats with CCI, near-perfect linear trends in mean power were observedfor iS1, PFC and cS1 (R²=0.96, 0.89, and 0.95, respectively), whereas anear-perfect polynomial trend was observed for PWL at the samelongitudinal time points.

The present EEG data reflect a spontaneous, 15 second interval duringresting state, whereas the behavioral data represent an evoked, pawwithdrawal reflex. Generally, an increase in EEG power correlated with adecrease in the latency of PWL. This relationship was consistent forcapsaicin and CFA conditions across waveforms recorded via all three EEGelectrodes, with the exception of iS1 after capsaicin, as discussedabove. Moreover, a longitudinal inverse plateau trend was observed inPWL, whereby values at d7, d14, and d16 after CCI were not statisticallydifferent, in contrast to the ascending linear trend over time for S1EEG mean power. Thus, EEG power provides valuable information regardingthe chronic nociceptive state, which cannot be inferred from solely PWL.

Example 6. Effect of Administering Analgesics on EEG Power

The sensitivity of EEG power to analgesic treatment was investigatedusing the clinically relevant drugs ibuprofen, pregabalin andmexiletine. Ibuprofen, a NSAID cyclooxygenase inhibitor, is widely usedas a non-prescription analgesic which was initially developed for mildforms of musculoskeletal and arthritis pain. Pregabalin, ananticonvulsant α2δ-subunit ligand, is clinically effective for themanagement of peripheral neuropathic pain and post-incisional pain, aswell as cutaneous and muscle hyperalgesia in inflammatory models ofmuscle pain. Mexiletine, a non-selective, use-dependent voltage-gatedsodium channel blocker (which is also anti-arrhythmic), has been shownto suppress persistent sodium currents in peripheral sensory axons ofpatients and is considered a third-line treatment for neuropathic pain.

For cS1, treatment with ibuprofen (FIG. 4A) did not have a significanteffect on EEG mean power (122±10, n=4 rats) compared to capsaicin alone(128±17; n=8 rats), whereas pregabalin treatment in rats with CFAreduced mean power from 299±70 to 209±35 (p<0.05, n=7 rats; FIG. 4A). Inrats with CCI, treatment with pregabalin or mexiletine reduced meanpower from 217±39 to 87±12 (p<0.05, n=5 rats) and from 245±56 to 134±18(p<0.05, n=5 rats), respectively. Similar results were observed for PFCand iS1.

For PFC, treatment with ibuprofen did not have a significant effect onEEG mean power (117±18, n=4 rats) compared to capsaicin alone (123±16;n=8 rats), whereas pregabalin treatment in rats with CFA reduced meanpower from 208±26 to 138±20 (p<0.05, n=5 rats). In rats with CCI,treatment with pregabalin or mexiletine reduced EEG mean power from188±27 to 133±14 (p<0.05, n=7 rats), and from 156±10 to 113±6 (p<0.05,n=6 rats), respectively. For iS1, treatment with ibuprofen did not havea significant effect on mean power (143±35, n=4 rats) compared tocapsaicin alone (124±14; n=8 rats), whereas pregabalin treatment in ratswith CFA reduced EEG mean power from 20824 to 133±9 (p<0.05, n=5 rats).In rats with CCI, treatment with pregabalin or mexiletine reduced EEGmean power from 219±32 to 151±20 (p<0.05, n=6 rats), and from 247±78 to13926 (p<0.05, n=8 rats), respectively.

The analgesic effect of these drugs was also tested behaviorally in thesame animals. Although ibuprofen had no effect on EEG mean powerfollowing capsaicin, ibuprofen blocked thermal hyperalgesia byincreasing PWL from 64±3 (n=8 rats) to 114±10 (p<0.05, n=4 rats; FIG.4B). Pregabalin also increased PWL in rats with CFA from 60±3 to 132±17(p<0.05, n=5 rats). In rats with CCI, treatment with pregabalin ormexiletine increased PWL from 81±3 to 119±5 (p<0.05, n=7 rats) and from83±5 to 119±17 (p<0.05, n=7 rats), respectively.

In summary, ibuprofen was effective in attenuating thermal hyperalgesia,but did not have a significant effect on EEG power, which could resultfrom the differential effects of the mechanism of action of ibuprofen onevoked versus spontaneous pain. Otherwise, pregabalin and mexiletineeffectively blocked thermal hyperalgesia and reversed EEG mean power tonormal levels in rats with CFA and CCI. These results further confirmthat pregabalin and mexiletine, at the optimal analgesic doses used inthis study, did not manifest adverse EEG signs, such as diffuse orparoxysmal slow activity that is often associated with drowsiness andwould have an enhancing effect on EEG power in the low-frequency range.

Example 7. Coherence of Brain Regions Following Pain

The effect of pain on cortico-cortical S1-PFC coherence was alsoinvestigated. Coherence in the 3-30 Hz range between cS1-PFC increasedin rats more than 14 days after CCI, whereas capsaicin and CFA did notcause a significant change in cS1-PFC coherence (FIG. 5A). Inparticular, coherence between cS1 and PFC (following values are mean3-30 Hz coherence) did not change in rats with capsaicin (0.60±0.06 innaïve and 0.61±0.05 in capsaicin; n=7 rats) or CFA (0.67±0.4 in naïveand 0.67±0.03 in CFA; n=11 rats). Similarly, cS1-PFC coherence was notchanged in rats at day 7 (d7) after CCI (0.68±0.05) compared to naïve(0.65±0.04; n=5 rats), whereas it was significantly (p<0.05) increasedstarting at day 14 (d14) following nerve injury (0.71±0.04; n=5 rats).Analgesic treatment with pregabalin or mexiletine reversed cS1-PFCcoherence (p<0.05), with mexiletine having a more pronounced attenuatingeffect (0.71±0.04 for CCI d14 and 0.61±0.03 after pregabalin and0.68±0.03 for CCI d16 and 0.50±0.10 after mexiletine, respectively; n=5rats). Similarly, capsaicin and CFA did not significantly effect cS1-iS1coherence and iS1-PFC coherence.

Coherence between iS1 and PFC was significantly (p<0.05) enhanced at d7(0.61±0.03; n=6 rats) and d14 (0.69±0.04 compared to naïve 0.59±0.06)after CCI (FIG. 5B.) Coherence between cS1 and iS1 was alsosignificantly (p<0.05) enhanced at d7 (0.63±0.06 in naïve compared to0.72±0.02 d7; n=6 rats) and d14 (0.63±0.06 in naïve compared to0.75±0.04 d14; n=6 rats) after CCI (FIG. 5C).

In summary, S1-PFC coherence is enhanced in rats at d14 after CCI,corresponding to a late-stage neuropathic pain. This result indicatesthat increased functional connectivity between S1-PFC may predict paintransition from an acute to a chronic stage. In contrast,inter-hemispheric coherence between iS1 and cS1 increases in rats withCCI as early as d7 after CCI.

Lastly, control experiments demonstrated that cS1 mean EEG power did notsignificantly changed in rats following capsaicin sham (93±31 versus100±24 in naïve; n=3 rats), d7 CCI sham (105±27 versus 100±24 in naïve;n=6 rats), and d2 CFA sham (85±11 versus 100±23 in naïve; n=5 rats; FIG.6). We also confirmed that intradermal vehicle injection in the lefthindpaw of rats at d2 after CFA had no effect on mean cS1 EEG power(94±4 compared to CFA d2 pre-vehicle 100±9; n=4 rats). Moreover,coherence in the 3-30 Hz range between waveforms recorded via pairs ofEEG electrodes did not change in these same control experiments.

In the present study, we used a relatively non-invasive EEG recordingmethods in awake, freely behaving rats to demonstrate that painmodulates on-going oscillations in clinically relevant models and thateffective analgesic drugs reverse this modulation. These results suggestthat brain oscillations predict spontaneous nociceptive states inrodents (FIG. 12).

Example 8. Increased PSD During Pain States in Humans

A wireless, 16-electrode, single-use EEG system (StatNet™; BioSignal)was used to determine waveforms in brain tissue of healthy humansubjects during a pain state of distress-tolerance (FIG. 7). Subjectswere randomized to receive ice water or room temperature water for 20second intervals. Subjects submerged a hand in the bucket of water (icewater or room temperature) and were then asked to rate their pain scoreat various times (FIG. 8A). Subjects who received ice water reported alower pain score during the first 10 seconds of submersion compared tothe last 10 seconds of submersion. There was an increase in power in thetheta range (6-7 Hz) associated with the higher pain score at the Fzplacement electrode (FIG. 8B). Source localization showed a prominent6-7 Hz increase in power corresponding to Fz, which overlaps withfrontal cortex in humans and PFC in rats (FIG. 8C). Subjects whoreceived room temperature water did not exhibit an increase in powercorresponding to the Fz and did exhibit decreased PSD across multiplefrequency bands (3-30 Hz) in caudal brain regions.

Example 9. Determination of Theta Oscillations in Somatosensory Cortexand Thalamic Bursts Following Pain

Experiments were performed to investigate the relationship between painbehavior, theta (4-8 Hz) oscillations in somatosensory cortex, and burstfiring in thalamic neurons in vivo. Thalamic bursts are triggeredpredominantly by GABAergic drive from the reticular thalamic nucleus(TRN), a thin layer overlaying the thalamus that receives strong inputfrom limbic cortical areas. To selectively induce thalamic bursts, TRNswere optically stimulated in awake, unrestrained transgenic miceco-expressing the vesicular GABA transporter (VGAT) withChannelrhodopsin-2 (ChR2). In these mice, ChR2 expression in thethalamus is restricted to the TRN. Age-matched wild-type (C57 Bl\6J)non-ChR2 expressing mice were also used to control for non-specificoptical stimulation effects. The naïve state refers to normal conditionsprior to induction of the pain models.

A custom-made multi-channel system was used to record extracellularlyfrom putative single-units in ventral posterolateral (VPL) thalamus andlocal field potential (LFP) in the primary somatosensory cortex (S1)hindlimb area (FIGS. 9A-9B). For a description of the FlexDrive systemassembly see www.open-ephys.org/flexdrive. Drives were positioned overthe right side of the brain targeting the VPL thalamus (Bregma −1.22 to−1.40, 1.75 to 2.00 lateral, 3 to 4 mm vertical) and SI cortex (Bregma−0.86 to −1.10, 1.5 to 1.8 lateral, <0.5 mm vertical). In each mouse,3-4 tetrodes were positioned in VPL or SI and one tetrode in TRN, wherean optical fiber was positioned over the somatosensory TRN (Bregma −1.20to −1.34, 2.30 to 2.40 lateral, 3.5 mm vertical). FlexDrives were fixedto the skull using C&B-METABOND® Quick! Adhesive (Parkell). After 3 dayspostoperatively, tetrodes were lowered incrementally (˜500 μm over 5-7days) until auditory confirmation of typical neuronal responses asexpected in VPL and SI (e.g., increased multiunit firing) evoked bylight brushing of the left hindpaw. Tetrode positions were alsocorroborated by stereotaxic coordinates. Additional criteria foridentifying VPL units included peak-to-trough duration of the actionpotential and the observations that most VPL neurons increase in firingrate in response to gentle brushing and noxious pinch of the receptivefield (i.e. wide dynamic range type) while TRN neurons are predominantlyinhibited. Chronic implants were stable over several weeks, allowinglongitudinal analysis of neuronal activity with behavioral testing ofmechanical sensitivity.

For electrophysiological recording in naturally behaving mice, mice werebriefly sedated (1% isoflurane <2 min) to allow connection of theFlexDrive to two-16 channels preamplifier (TDT RA16PA), headstages (TDTLP16CH) and a fiber optic patch cord (200 μm). Unrestrained mice laterrecovered from sedation in a 3×3″ PLEXIGLAS® enclosure for at least 15minutes prior to the start of electrophysiological recording using a TDTRZ2 BioAmp processor at 24.4 kHz sampling rate per channel. Twosequential notch filters (58-62 Hz) were applied to reduce electricalinterference. The behavior of the animal was noted to determine alertrest periods, defined as alertness with no vigorous movements such asgrooming or scratching. At the end of the final recording session,electrolytic lesions were performed for postmortem histologicalverification of recording sites, whereby brains were removed,immediately placed in cryogenic compound (OCT), and frozen at −80 C forfurther cryosectioning. Serial sections (25 μm) were treated with cresylviolet and hematoxylin for viewing under light microscope.

For tonic and burst spike sorting, extracellular spike waveforms (actionpotentials) in VPL were detected and sorted from LFP waveforms, bandpassfiltered at 300-3000 Hz, using primarily template matching and principlecomponent algorithms in Spike2 (CED 1401, Cambridge Electronic Design,UK). Sorted spikes were then screened visually and inspected forfalse-positive or overlapping unitary assignments. Only one electrodeper tetrode was used for spike sorting to minimize redundant assignmentsfrom the same unit. Hence, 3-4 units were isolated from VPL per mouse,whereas cortical oscillations reflected the mean of 3-4 LFP measurementsin SI. Moreover, isolation of putative unitary spikes also met thecriterion of inter-spike interval (ISI)>2 ms (refractory period). Burstanalysis was performed on sorted spikes and others related to thalamicbursting evoked specifically by TRN stimulation, whereby burst eventswere identified according to the following parameters: maximum intervalsignifying burst onset=4 ms, offset=8 ms, longest increase in ISI withina burst=2 ms, and minimum number of spikes within a burst=2.

For optical stimulation of TRN, laser light pulses were generated usinga 100 mW 473 nm laser (MBL473 OptoEngine LLC) connected to the FlexDrivevia fiber patch cord. Pulse control was achieved using an isolatedpulsegenerator (A-M systems 2100) at a 10 Hz frequency, 0.5 ms pulsewidth, and total duration of 5 sec during electrophysiologicalrecording. For behavioral testing of the mechanical withdrawalthreshold, optical stimulation was applied for 2 seconds during theapplication of von Frey filaments.

For acute and chronic pain models, capsaicin (0.1%, 10 μl, intradermal)was injected into the plantar aspect of the left hindpaw under sedation(1.5% isoflurane <2 min) to prevent stress due to restraining thehindpaw. A TRPV1 agonist, capsaicin causes increased neuronal firing ofnociceptors, mainly polymodal C-fibers. Chronic constriction injury(CCI) was induced in the same mice that underwent capsaicin treatment at3 days post-injection after verifying that mechanical withdrawalreturned to normal. The sciatic nerve was exposed unilaterally afterskin incision at the midthigh level and blunt dissection of the bicepsfemoris under deep anesthesia (isoflurane, 3.5%). Four chromic gut (5-0)ligatures were tied loosely around the nerve 1 mm apart and theoverlying muscles and skin were closed in layers with 5-0 ETHILON®sutures.

Fast Fourier transform function (FFT) was used to convert LFP waveformfrom the time domain to the frequency domain, yielding power spectraldensity (PSD) histograms using 5 sec time intervals during awake restingstate (no difference was found compared to the multi-taper method).Values were generated at 57 frequencies (0.47 Hz bins) between 3-30 Hz.For the pain state, data were collected within 15-20 min after capsaicininjection.

Mechanical sensitivity of the hindpaw was assessed by measuring thethreshold of withdrawal in response to the application of calibrated vonFrey filaments of different bending forces to the plantar aspect of thehindpaw according to the ‘up-down’ method, whereby filaments ofdifferent bending forces were pressed against the paw until buckling fora maximum of 3 seconds or a withdrawal reflex. This test representsnaturally-occurring stimulation to the hindpaw in the noxious andnon-noxious range evoking a biologically-relevant state in mammals.

In the dual chamber conditioned place preference (CPP) test,FlexDrive-implanted mice were conditioned with unrestricted access toboth chambers for three days, with baseline preference determined on thethird day. On the fourth day, mice underwent ‘pairing’ by beingindividually restricted to one chamber and receiving optical stimulation(10 Hz, 0.5 ms pulse width) for 30 sec, then 4 hours later they wererestricted to the opposite chamber for 30 min after receivingoptogenetic stimulation. On the fifth day, mice were allowed free accessto both chambers. Chamber preference was video recorded and analyzedoff-line by an observer blinded to the animal's treatment.

The distribution of the number of bursts and spikes in VPL per bin, andthe magnitude of SI theta power per bin were analyzed for 919 bins foreach mouse (n=5, bin size 30 ms). Regarding SI theta power, the meanobserved power of 3 consecutive bins was used as the representativepower of a bin (e.g. the average of the observed power of the bini−1,the bini, and the bini+1 was used as the representative power of thebini) to satisfy the conditions of accurate power estimation (100 ms binsize) and fine temporal resolution (30 ms bin size). Analysis revealedthat both the number of bursts and spikes per bin had Poissondistribution and more than one burst or two spikes per bin wereconsidered significant events. SI theta power per bin had a lognormaldistribution.

The relationship between fluctuation of SI theta power and spikes orbursts was analyzed using cross-correlation analysis as describedpreviously. Briefly:Q(t)=1/(T−t)Σ_(i=1) ^(T-t) X(i)Y(i+t)

Where, in the case of burst, X(i) was 1 (if there were any bursts in thebini) or 0 (if there was no burst in the bini), and in the case ofspikes, X(i) was 1 (if there were more than two spikes and no burst inthe bini) or 0 (otherwise). Y(i+t) represented the fluctuation of thetapower with t bins lags from the bini, and was calculated as follows:Y(i)=df(t)/di={f(i+1)−f(i)}/Δt

Where f(i) represents “−log transformed S1 theta power at bini”, and Δiis the size of bini. If no relationship is found between bursts orspikes in VPL and fluctuation in SI power, Q(t) would have normaldistribution. Thus, Z value was calculated for each Q(t) as follows:Z(t)={Q(t)−E[Q(t)]}/√{square root over (V[Q(t)])}Where:E[Q(t)]=E[X)E[Y]AndV[Q(t)]=1/(T−t)(E[X ²]E[Y ²]−E[X]² E[Y]²)

Z(t) was calculated for each mouse, and then, the average of Z(t) andthe 95% confidence interval of Z(t) were calculated.

Analysis of variance (ANOVA) and parametric tests were used forstatistical analysis. Two-way ANOVA analysis followed by Bonferroni'scorrection, Student's t-test, or the z-score method was used to computestatistical significance. Bartlett's test was performed to computenormal distribution and equal variance. A P value <0.05 was consideredsignificant (denoted with * in figures). For behavioral and power data,comparisons were made between animal groups and for spike and burstactivity data comparisons were made between neuronal groups. All valuesare reported as ±standard error of the mean (SEM).

Example 10. Thalamic Bursts Down-Regulate Cortical Theta and NociceptiveBehavior

Histological analysis confirmed that ChR2 expression was limited toGABAergic neurons in the TRN (FIGS. 9C-9D). Optical stimulation at 10Hz, which is consistent with the physiological ‘baseline’ firing rate ofTRNs, effectively reduced SI theta power in sedated mice (FIG. 10A). Inparticular, TRN stimulation at 0.5, 10, and 50 Hz reduced SI theta powerto 5.14×10⁻²±710×10⁻² mV², 4.35×10-2±0.33×10⁻² mV², and4.45×10-2±0.36×10⁻² mV², respectively, compared to the baseline SI thetapower of 5.48×10⁻²±0.38 mV². TRN stimulation at 10 Hz reduced power inawake, resting mice within the theta range of 3.8-8.5 Hz from4.70×10⁻²±0.25×10⁻² mV² to 3.97×10⁻²±0.40×10⁻² mV² (FIG. 10B; P=0.033).Moreover, TRN stimulation increased the burst firing rate of putativesingle-units in VPL from 0.07±0.09 Hz to 1.01±0.31 Hz (FIG. 10C,P=0.002). This stimulation also enhanced the threshold of paw withdrawalto von Frey stimuli from 3.38±0.52 g to 5.02±0.88 g (FIG. 10D; P=0.03).These results show that rescue of TRN function by selective opticalstimulation releases thalamic neurons from inhibition, and thus,promotes thalamic bursting, reduces cortical theta, and reversesnociceptive behavior.

Next, the effect of TRN stimulation on thalamic firing was investigatedin a pain model. SI power increased significantly in the theta bandwithin 15-20 minutes after intradermal injection of capsaicin in thehindpaw from 4.82×10⁻²±0.57×10⁻² mV² to 8.15×10⁻²±0.13×10⁻² mV² at3.8-6.2 Hz (FIG. 11A; P=0.048). In these mice, TRN stimulationeffectively reversed the pain-related increase in SI power to normallevels from 8.15×10⁻²±0.13×10⁻² mV² to 4.55×10⁻²±0.75×10⁻² mV² (FIG. 3a; P=0.002). The rate of spontaneous burst firing in VPL neurons(0.02±0.02) increased after capsaicin injection (0.64±0.11) and wasfurther enhanced during TRN stimulation (1.50±0.25) in the same neurons(FIG. 11B; *P=0.00002, *P=0.001). Paw withdrawal threshold decreasedwithin 15-20 minutes after capsaicin injection from 4.47±1.07 g to.1.00±0.37 g suggesting mechanical allodynia, which is a hallmark ofneuropathic pain (FIG. 11C; *P=0.011). Optical TRN stimulation, however,elevated withdrawal thresholds to near pre-capsaicin levels of 4.28±1.22(FIG. 11C; *P=0.023). Reversal of these anti-nociceptive effects to1.41±0.22 occurred within 5 min afterwards (FIG. 11C; *P=0.048). Wefurther investigated the longitudinal effects of TRN stimulation onthalamic firing, theta power, and nociceptive behavior following chronicconstriction injury (CCI) of the sciatic nerve in the same animals. Theresults of these studies are comparable overall to those obtained in thecapsaicin pain model.

The temporal relationship between thalamic firing and cortical theta wasthen investigated. As shown in a representative example of a time seriesof SI spectrogram with corresponding VPL burst rate, epochs of hightheta power and burst events do not coincide temporally (FIG. 11D).Dynamic, time-lagged cross-correlation between burst or tonic firingrate versus theta power revealed a significantly negative correlationbetween theta amplitude and burst rate, which suggests that bursts (butnot tonic firing) likely trigger the down-regulation of SI theta with atime lag of 120 ms (FIG. 11E).

Promotion of burst firing in thalamocortical neurons during naïve andpain states is negatively correlated with cortical theta and mechanicalallodynia. Our data show that optogenetically-induced thalamic burstsattenuate pain-induced cortical oscillations and enhance withdrawalthreshold to mechanical stimuli. These results indicate that thalamicbursts are an adaptive response to pain that de-synchronizes corticaltheta and decreases sensory salience. Optogenetic stimulation of thethalamic reticular nucleus promotes burst firing in the thalamus whiledown-regulating theta oscillations in the somatosensory cortex andattenuating pain behavior.

OTHER EMBODIMENTS

Various modifications and variations of the described methods will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it will be understood that itis capable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention that are obvious to those skilled in the art are intended tobe within the scope of the invention. This application is intended tocover any variations, uses, or adaptations of the invention following,in general, the principles of the invention and including suchdepartures from the present disclosure come within known customarypractice within the art to which the invention pertains and may beapplied to the essential features herein before set forth.

What is claimed is:
 1. A method for detecting pain in a subject, themethod comprising: (a) recording theta or gamma waveforms in asomatosensory cortex or a frontal cortex of the subject byelectroencephalography (EEG); (b) applying fast Fourier transfer (FFT)to convert the waveforms from the time domain to the frequency domain,thereby producing power spectral density (PSD); and (c) determiningpower amplitude from the PSD, wherein an increase in the power amplitudein a theta or gamma frequency band in the somatosensory cortex or thefrontal cortex relative to baseline serves as an indicator of pain.
 2. Amethod for detecting pain in a subject, the method comprising: (a)recording theta or gamma waveforms in a somatosensory cortex or afrontal cortex of the subject by EEG; (b) applying FFT to convert thewaveforms from the time domain to the frequency domain, therebyproducing PSD; (c) determining power amplitude from the PSD; and (d)determining coherence of brain regions from the FFT, wherein an increasein the power amplitude in a theta or gamma frequency band in thesomatosensory cortex or the frontal cortex relative to baseline and anincrease in the coherence of brain regions serve as an indicator ofpain.
 3. The method of claim 2, wherein the coherence of brain regionsis determined from the difference in coherence at individual frequencyunits or frequency bands.
 4. The method of claim 1, further comprisingstimulating a thalamic reticular nucleus (TRN) of the subject if anincrease in power amplitude is detected.
 5. The method of claim 4,wherein the TRN is stimulated with a laser-emitting optic fiber adaptedfor implantation in the brain of the subject or a therapeutic agent. 6.The method of claim 1, wherein the waveforms are theta waveforms.
 7. Themethod of claim 1, wherein the waveforms are gamma waveforms.
 8. Themethod of claim 1, wherein the waveforms are theta and gamma waveforms.9. The method of claim 1, wherein step (a) comprises recording in thesomatosensory cortex.
 10. The method of claim 1, wherein step (a)comprises recording in the frontal cortex.
 11. The method of claim 1,wherein step (a) comprises recording in the somatosensory cortex and thefrontal cortex.
 12. The method of claim 1, wherein the power amplitudefrom the PSD is determined in the theta frequency band.
 13. The methodof claim 1, wherein the power amplitude from the PSD is determined inthe gamma frequency band.
 14. The method of claim 1, wherein the poweramplitude from the PSD is determined in the theta and gamma frequencybands.
 15. The method of claim 1, wherein the power amplitude from thePSD is determined in the somatosensory cortex.
 16. The method of claim1, wherein the power amplitude from the PSD is determined in the frontalcortex.
 17. The method of claim 1, wherein the power amplitude from thePSD is determined in the somatosensory cortex and frontal cortex.